Bovine Leukemia Virus (BLV) is an oncogenic retrovirus responsible for the enzootic form of bovine lymphosarcoma, the most frequent malignancy of domestic cattle (Ferrer (1980 Adv. Vet. Sci. Comp. Med. 24:1–68). BLV infection results in a 1–8 year long asymptomatic period (Ferrer et al. (1979) J. Am. Vet. Med. Assoc. 175(7):705–8), followed by development of persistent lymphocytosis (PL) in approximately 30% of infected cattle with progression to a malignant lymphosarcoma in fewer than 10% of the animals (Ferrer et al. (1979) J. Am. Vet. Med. Assoc. 175(7):705–8). The PL stage is a benign neoplasia of B lymphocytes, which are the predominant or exclusive targets of BLV (Esteban et al. (1985) Cancer Res. 45(7):3225–30). This stage of infection is associated with an increased percentage of peripheral B lymphocytes containing provirus as well as increased viral gene expression (Mirsky et al. (1996) J. Virol. 70(4):2178–83). The development of PL markedly enhances the probability of transmission (Mammerickx et al. (1987) Leuk Res. 11:353–58). The critical importance of PL to transmission of this blood-borne disease was demonstrated by experiments showing that it required significantly less blood from cattle with persistent lymphocytosis to transmit BLV than blood from infected cattle which did not have persistent lymphocytosis (Mammerickx et al. (1987) Leuk. Res. 11:353–58). Moreover, vertical transmission from BLV-infected dams to their calves has been shown to be strongly correlated with persistent lymphocytosis (Agresti et al. (1993) Amer. J. Vet. Res. 54:373–78).
In cattle, the ability to transmit BLV varies (Weber et al. (1983) Amer. J. Vel. Res. 44:1912–15); Marnmerickx et al. (1987) Leuk. Res. 11:353–58), and expression of antigen after in vitro culture has been shown to correlate with infectivity (Miller et al. (1985) Amer. J. Vet. Res. 46:808–13). The level of BLV expression in the animal also may correlate with the probability of development of persistent lymphocytosis (Cockerell et al. (1988) Leuk. Res. 12:465–69; Dropulic et al. (1992) J. Virol. 66:1432–41). Moreover, persistent lymphocytosis is a strong risk factor for development of lymphoma. In 1–10% of the animals with persistent lymphocytosis, B cell clones undergo neoplastic transformation, leading to leukemia or lymphoma, and cattle with persistent lymphocytosis are three times more likely to develop lymphoma than infected cattle without persistent lymphocytosis (Ferrer et al. (1979) J. Am. Vet. Med. Assoc. 175(7):705–8).
BLV is prevalent in dairy operations, with up to 89% of the U.S. dairy operation seropositive for BLV (Howie (1997) Feedstuffs 69:11). Not only does the virus kill cattle, milk and fat yields in BLV-infected cows with persistent lymphocytosis are greatly reduced (Da et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6538). Moreover, BLV also produces malignant lymphomas in sheep (Wittman et al. (1989) Arch. Exp. Veterinaermed. 23:709). However, the greatest economic impact of BLV infection in the United States arises from the fact that several countries will not import cattle from BLV-infested areas. Although various attempts have been made to develop a vaccine against BLV infection, an effective vaccine to protect cattle or sheep is not available (Miller et al. (1978) Annales de Recherches Veterinaires 9:871; U.S. Pat. No. 4,323,555). BLV infection is thus a costly impediment to cattle production.
Peripheral blood mononuclear cells (PBMC) from BLV-infected cattle proliferate spontaneously in vitro (Takashima & Olson (1981) Arch. Virol. 69(2):141–8; Thorn et al. (1981) Infect. Immun. 34(1):84–9). This spontaneous lymphocyte proliferation (SLP) is particularly vigorous in PBMC cultures from cattle in the PL stage of infection. Derepression of viral gene transcription and the synthesis of viral proteins precede SLP (Kettmann et al. (1976) Proc. Natl. Acad. Sci. USA. 74(4)1014–18; Baliga & Ferrer (1977) Proc. Soc. Exp. Biol. Med. 156(2):388–91; Ferrer (1980) Adv. Vet. Sci. Comp. Med. 24:1–68). Therefore, SLP provides a tractable model system for identifying factors that are capable of preventing BLV-induced neoplasia and malignant lymphoma in infected cattle. The present invention shows that Shiga-toxin type 1 (Stx1) is a potent and selective inhibitors of BLV-induced SLP. The present invention also shows that Shiga-toxin type 2 (Stx2) is a potent and selective inhibitors of BLV-induced SLP.
The family of Shiga toxins includes Stx type 1 (Stx1), Stx type 2 (Stx2) and Stx type 2 variants (Donahe-Rolfe et al. (1991) Rev. Infect. Dis. 13(Suppl. 4):S293–7). These toxins belong to a large family of ribosome-inactivating proteins (RIPs) that are found in a variety of higher plants and some bacteria. Most RIPs are hemitoxins (enzymatically active A chains) and some are holotoxins (one A chain associated with a specific number of B changes. Thus, class 1 RIPs (hemitoxins) are N-glycosidases that inactivate ribosomes by removing a single adenine in a specific ribosomal RNA sequence (Endo et al. (1987) J. Biol. Chem. 262:5908–12; Endo et al. (1988) Europ. J. Biochem. 171:45–50). Class 2 RIPs (holotoxins) are composed of an A subunit homologous to class 1 RIPs, noncovalently joined to one or more B subunits, usually galactose-specific lectins, that facilitate toxin binding and uptake into target cells. Holotoxins are highly toxic to cells expressing receptors for B subunit(s), but not to receptor-deprived cells, and are not toxic to normal cells as isolated A chains (Barnett et al. (1991) Antiviral. Res. 15:125–38; Dosio et al. (1994) J. Pharm. Sci. 83:206–11; Girbes et al. (1996) Cell. Mol. Biol. (Noisy-le-grand) 42:461–71). Plant hemitoxins are not toxic to the plants that synthesize them and have low cytotoxicity against animal cells, unless the cells have high pinocytic activity (Change et al. (1979) Contraception 19:175–84; Yeung et al. (1988) Int. J. Pept. Protein Res. 31:265–8).
Plant RIPs of both class 1 (e.g., pokeweed antiviral protein, titrin, trichosanthin) and class 2 (e.g., ricin) have potent antiviral activities (Stirpe et al. (1992) Biotechnology (NY) 10:405–12). These compounds often inhibit viral proliferation in mammalian cells in vitro, and some have been tested in vivo in clinical or laboratory settings. For example, plant hemitoxins can enter and eliminate virally-infected plant cells, and some are also found to be highly toxic to various virally-infected animal cells (Girbes et al. (1996) Cell Mol. Biol. (Noisy-le-grand) 42:461–71). The class 2 RIP ricin can eliminate latent herpes simplex virus in mice (Hino et al. (1988) J. Infect. Dis. 157(6):1270–1). Other plant RIPs inhibit replication of human immunodeficiency virus type 1 (HIV1) in human peripheral blood mononuclear cells at concentrations nontoxic to uninfected cells (Olson et al. (1991) AIDS Res. Hum. Retroviruses 7(12)1025–1030; Lee-Huang et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92(19):8818–22).
Shiga toxins are class 2 RIPs composed of an A subunit associated with a pentamer of receptor-binding B subunits. Because of their ability to bind to target cells, class 2 RIPs are potent cytotoxins. Stx1 is toxic to cells that express high levels of the toxin receptor, globotriosylceramide (Gb3 or CD77), most notably Vero cells and human glomerular endothelial cells (Jackson (1990) Microbial. Pathogenesis 8:235–42).
One problem associated with using RIPs as general antiviral agents is their specificity (Wachinger et al. (1993) Res. Exp. Med. 193(1):1–12; Watanabe et al. (1997) Biosci. Biotechnol. Biochem. 61:994–997). For example, the RIP Bryodin selectively inhibits the growth of HIV-1-infected cells, whereas RIPs gelonin and ricin did not (Wachinger et al. (1993) Res. Exp. Med. 193(1):1–12). Another concern is that RIPs are highly cytotoxic (Benigni et al. (1995) Int. J. Immunopharmacol. 17:829–39; Sparapani et al. (1997) Glia 20:203–9; Yoshida et al. (1999 J. Infect. Dis. 180:2048–52). Therefore, although the antiviral effects of some RIPs are known, the use of RIPs as antiviral agents has not been generally applicable. Surprisingly, it has been discovered that Stx1 strongly inhibits BLV-related cell proliferation and BLV expression and does not cause indiscriminate cell death (Ferens & Hovde (2000) Infect. Immun. 68:4462–9). Specifically, this activity is manifested by subunit A of Shiga-toxins, which is nontoxic to ruminants or humans.